Remediation of contaminates including low bioavailability hydrocarbons

ABSTRACT

A method of treatment of a contaminated material contaminated with an organic compound. The method includes treating the contaminate with a chemical oxidation step and a bioremediation step. The chemical oxidation step includes treating the contaminate with the following: a transition metal in soluble form in combination with an isolated chelator of the transition metal, to form a transition metal:chelator complex; an oxidizing agent that provides a reactive free radical in the presence of the transition metal complex; and a buffering compound; the pH being maintained in a neutral range. The method of treatment further includes treating the contaminate with a microbial consortium including at least two species prior to or after the chemical oxidation step to enhance the remediation process.

RELATED APPLICATIONS

This application is a continuation of U.S. Pat. No. Ser. No. 09/863,491filed May 23, 2001 now claims the benefit of U.S. Pat. No. 6,623,211,which claims benefit of U.S. Provisional Application Serial No.60/206,703 entitled: “COBR: Combined Oxidation and Biotreatment forRemediation of Soils Contaminated with Low BioavailabilityHydrocarbons,” filed May 23, 2000.

This work was supported by a grant (Project SITE-57) from the New JerseyHazardous Substance Management Research Center and was funded in part bya grant from the National Institutes of Environmental Health Sciencesthrough Superfund Basic Research Program Grant P42-ES-04911. Thegovernment may have certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to the field of remediation of chemicalcontamination, including for example, contamination in particulates suchas soil and also in fluids such as groundwater. More particularly, thepresent invention relates to a method of efficiently treatingparticulates and fluids contaminated with organic materials, such ashydrophobic aromatic hydrocarbons using biocompatible methods foroxidation of the organic contaminants.

BACKGROUND

Chemical contamination of the environment, particularly of soil andgroundwater is currently a widespread problem that is prevalent in manyparts of the industrialized world. Industrial pollution has contaminatedmillions of acres of soil and associated aquifers. Often, cleanup of thecontamination is avoided because of the costs of remediation, and theland may remain unused or abandoned.

Typical remediation (decontamination) strategies include incinerationand/or removal. In the case of contamination of large areas, an on-siteincinerator may be warranted. In other cases, generally on a smallerscale, excavation and removal to an RCRA (Resource Conservation &Recovery Act) compliant incinerator or landfill may be employed. Both ofthese methods require intensive labor and mechanical effort resulting inhigh costs. Other methods in frequent use include pumping and treating,vacuum extraction, steam flooding, air sparging and soil flushing.

Treatment of contaminated soils in situ by various methods has beenpursued because it does not require excavation or hauling and is lesscostly. Oxidation is one technique used to treat contaminated soils insitu. For example, one such oxidation technique that has been widelyused is oxidation of organic compounds with ozone, potassiumpermanganate or hydrogen peroxide.

Additionally, efforts have been made to reduce the costs of treatmentwith chemicals by more effectively directing the chemicals used in thetreatment to the contaminated soil to the appropriate location anddepth. For example, U.S. Pat. No. 5,525,008 to Wilson discloses a methodof directing the flow of the oxidizing treatment solutions tocontaminants in the soil or groundwater. The invention discloses the useof multiple horizontally spaced sealed injection wells, with the goal ofdirecting the reactive solution through the contaminated area.

Another approach at directing treatment chemicals to the contaminates insoil is disclosed by U.S. Pat. No. 4,591,443 to Brown. The '443 patentdiscloses a method of decontaminating subterranean soil by controllingthe mobility of aqueous treatment fluids. In order to direct thetreatment fluid containing the active chemical, such as hydrogenperoxide to the intended reaction site, hydratable polymers are used asviscosity modifiers. Optionally, cross linking agents may be added tofurther increase the viscosity of the treatment fluid. Surfactants areemployed to decrease the interaction of metals or clays with peroxide.The '443 patent further discloses the use of peroxide stabilizers, freeradical initiators such as iron (Fe) and also free radical inhibitors.Penetrating pre-treatment fluids for altering the reactivity of the soilor rock formation and inactivating hydrogen peroxide decompositioncatalysts are also disclosed.

The '443 patent teaches that these various oxidation and flow modifierscombine to provide a degree of spatial and temporal control of theoxidizing treatment chemicals, with the stated intention of reactingwith the desired chemical contaminants rather than the surroundingnaturally occurring minerals and soil. The '443 patent does not addressthe use of bioremediation, to treat many common forms of contamination,particularly organic contaminants such as polycyclic aromatichydrocarbons (PAHs), thereby permitting reduced chemical exposure andloading of the site. The '443 method would be expected to produce pooroxidation of, e.g. anthracene and chrysene, and other such hydrocarbons.

Bioremediation has begun to gain wider acceptance as a viable treatmenttechnology for remediating soils, sediments and subsurface sitescontaminated with hydrocarbons. The attractiveness of bioremediationarises at least in part from the fact that the process takes advantageof intrinsic biodegradative processes of microorganisms and because thecompounds that are the target of remediation are degraded to innocuousend products. In this respect, bioremediation-based remediationapproaches using either in situ or off site designs have beensuccessfully employed for remediation of soils and subsurface sitescontaminated with lighter fractions of petroleum or petroleum products,and for the lower molecular weight and more water soluble aromaticcomponents of petroleum products, represented, for example, by benzene,toluene, ethylbenzene and xylenes.

Bioremediation strategies, however, often have limited applicabilitywhen soils, sediments and subsurface sites are contaminated with complexmixtures of highly hydrophobic aromatic compounds such as commonlyoccurs for instance with tar residues. The polycyclic aromatichydrocarbons (PAHs) that are component of tar residuals, remain achallenge for the application of in situ remediation strategies owing tothe low aqueous solubility of mixed PAH components. Such PAHs areproduced, for example, from the volatile components of bituminous coalsin coal carbonization, from the residue of gasifying oils in oil gasprocesses, and from the cracking of enriching oils in carbureted watergas production at former manufactured gas plant (MGP) sites.Bioremediation of such PAH-contaminated sites is hampered by the lowaqueous solubility of PAH compounds, which leads to low bioavailabilitywhere the compounds are not available for microbial action that dependson aqueous chemistry and enzyme action.

Hydrogen peroxide, in the presence of ferrous ions (Fe⁺⁺) as a catalyst,generates a strong nonspecific oxidant hydroxyl radical that reacts withmost organic compounds at diffusion-controlled rates of 10⁷ to 10¹⁰ M⁻¹sect⁻². This is known as Fenton's reaction and has been used for thedestruction of organic contaminants including (poly)chlorinated aromaticcompounds and a variety of herbicides in aqueous solutions or soils.However, little evidence is available regarding whether the Fenton'sreaction can mineralize organic contaminants, or whether the resultingpartially oxidized organic compounds pose less hazards than the parentcompounds. Moreover, use of Fenton's reaction produces soil pH changeswhich are incompatible with bacteria and make subsequent use ofbioremediation methods ineffective.

Although Fenton's reagent has the potential to non-specifically oxidizemany PAHs, it also results in a substantial lowering of the soil pH,e.g. to a pH of between 2 and 3. At these pH levels, many heavy metalcontaminates become solubilized and migrate into ground water. Moreover,this pH range is subsequently incompatible with many forms of biologicaltreatment.

As noted above, one serious disadvantage of the use of chemicaloxidation with bioremediation is the lowering of soil pH to levels whichsolubilize many heavy metals and which are unacceptable for sustainingmany useful bacteria. Nonetheless, various attempts at treatingcontaminated soils have included a combination of bioremediation andchemical treatment.

U.S. Pat. No. 5,955,350 to Soni et al. discloses the stepwise use ofbiological treatment, then chemical treatment followed by anotherbiological treatment of organic waste. Hydrogen peroxide is a strongoxidant and is very reactive. The '350 patent discloses the use ofFenton's reagent and peroxide between two stages of biologicalremediation.

One severe drawback of the use of Fenton's reagent is that in somecircumstances, the rapid reaction of peroxide can result in excessiveheat and consequent generation of steam, creating high pressures andpotentially resulting in an explosive release. In the field, variousapproaches to the problem of explosive potential are used. These includeadaptations such as venting the formation or utilizing a slowintroduction of the peroxide. The '350 patent discloses a slow rate ofaddition of peroxide in order to avoid high rates of oxidation.Manageable temperatures are maintained by slow addition of hydrogenperoxide, exemplified by the addition of an approximate rate of 1 to 100mg hydrogen peroxide per hour per gram of contaminated soil to theferrous salt solution.

U.S. Pat. No. 5,610,065 to Kelley et al. also discloses combinedchemical and biological remediation including the use of Fenton'sreagent for degradation of high molecular weight PAHs in soil. The '065patent is silent as to pH control during the oxidation process. It isnot surprising, therefore, that the '065 patent follows chemicaloxidation with additional microbial inoculation in an effort to restockthe microorganism population after exposure to the harsh oxidationconditions. The '065 patent also discloses the use of a lower alcohol toincrease the aqueous solubility of PAHs.

U.S. Pat. No. 5,741,427 to Watts utilizes stabilizers to providechemical ligands for Fe(III) species during Fenton's oxidation. Thestabilizer ligands are provided by phosphates, silicates, or citrates.Control of pH is not addressed. Similarly, the aforementioned problemsassociated with oxidation of soil contaminants are not addressed.

To date the known methods which employ oxidation and/or bioremediationtechniques to treat contaminated soils all suffer from the variousdisadvantages discussed above. It would therefore be desirable toprovide a means by which oxidation of contaminates can occur efficientlywithout the need for undesirable changes in pH. There is a need for aremediation method that is operational in a neutral pH range withincreased biocompatibility and that reduces the solubilization of heavymetals. It would also be desirable to provide a treatment method whichavoids further contamination due to the formation of unwanted or toxicby-products as a result of the treatment.

SUMMARY OF THE INVENTION

The invention provides a method of treatment of a contaminated material,herein referred to as a contaminate, contaminated with an organiccompound. The method includes the steps of: providing a contaminate thatis contaminated with an organic compound, and treating the contaminatewith a chemical oxidation step. The chemical oxidation step includes:contacting the contaminate with a transition metal in soluble form; anda chelator of the transition metal, (such that the chelator of thetransition metal and the transition metal form a transitionmetal:chelator complex); and an oxidizing agent that provides a reactivefree radical in the presence of the transition metal complex; and abuffering salt to maintain the pH in the neutral range. The methodprovides a reactive free radical that initiates a chemical reaction withthe organic compound to produce reaction products of the organiccompound.

The method of treatment may further include pre-treating the contaminatewith a biodegradation step prior to or after the chemical oxidationstep. The biodegradation step includes a step of contacting thecontaminate with a microbial consortium under conditions suitable forthe consortium to mediate solubilization or biodegradion of the organiccompound.

The invention also provides a kit for treatment of a contaminatecontaminated with an organic compound, the kit comprising: (i) atransition metal in soluble form; (ii) a chelator of the transitionmetal that has the property of forming a transition metal:chelatorcomplex with the transition metal; and (iii) and a buffering salt tomaintain the pH in the neutral range. The kit may further comprise amicrobial consortium having the property of solubilizing or biodegradingan organic compound contaminant.

The invention further provides a method of producing a reactive freeradical in an aqueous medium in a neutral pH range, by providing atransition metal in soluble form in an aqueous medium, along with achelator of the transition metal, such that the chelator of thetransition metal and the transition metal form a transitionmetal:chelator complex; a buffering salt is optionally included tomaintain the pH in a neutral range with an oxidizing agent that providesa reactive free radical in the presence of the transition metal:chelatorcomplex.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have discovered methods that are safer, moreeffective and less expensive than the known methods for degradingorganic contaminants present in particulate materials or in liquidwastes. The methods are also effective for the degradation ofpollutants, the reduction of toxicity, the reduction of chemical orbiological oxygen demand and the removal of odors or color of acontaminate. The method involves oxidation of the contaminant compoundsby chemical treatment with optional biodegradation steps. These methodsare particularly effective in remediating organic contaminants,especially hydrocarbons that may have low solubility and lowbioavailability. Such contaminants are poorly if at all transformed bynatural processes alone.

Particulate materials such as soils and solid waste materials includefor instance, gravel, pebbles, stone, stone chips, rock, ore, miningwaste, coal, coke, slag, concrete, brick, construction material,demolition material, vermiculite, synthetic resin or plastic. Liquidwastes, include for example, industrial effluents in pools or holdingponds, contaminated ground water, liquid sludge as well as less pollutedaqueous run off. Such contaminated particulates, solids and liquids, arehereinafter interchangeably referred to as contaminates. Preferably thecontaminate is soil or ground water.

As used in the present specification the term “contaminate” refers toany matter containing an undesirable chemical component or pollutant.Examples of such undesirable chemical components or pollutants include,for instance, toxic chemicals, carcinogens, organic compounds,polycyclic compounds, aromatic compounds, aliphatic compounds, olefiniccompounds, ethynic compounds, acids, bases, alcohols, dyes, oils and thelike.

Among the contaminants that may be addressed by the methods of thepresent invention are the following listed for exemplification only andare in no way to be considered as limiting: hydrocarbons, polyaromatichydrocarbons (PAHs); dense non-aqueous phase liquids (DNAPLs) and lightnon-aqueous phase liquids (LNAPLs); solvents, particularly chlorinatedsolvents; tars and creosote; petroleum products and byproducts such as:any one or combination of the following—benzene, toluene, ethyl benzeneand xylene (known as BTEX), PAH, TPH and diesel fuel; chlorinatedhydrocarbons such as vinyl chloride, trichloroethylene (TCE),tetrachloroethylene, TCA, DCA, PCA and PCBs; chlorinated dioxins anddibenzofurans; phenolics; preservatives; pesticides; explosives andunspent munitions.

As used in the present specification the term “the neutral range” usedin reference to a pH range refers to a pH in a range around the neutralpH, which is pH of about 7. Preferably the pH is in a range from aboutpH 5 to about pH 8. More preferably the neutral pH range is from aboutpH 5.5 to about pH 7. Optimally the neutral pH range as referred toherein is from about 6 to about 6.5.

As used herein the term “buffering salt” means any salt maintains the pHin the neutral range. Preferably the buffering salt is soluble in acidicsolution. The buffering salt may have a pK_(a) in the range of about 5to about 8. A preferred buffering salt is calcium carbonate (CaCO₃).

In one aspect the present invention provides a method of treatment oforganic compounds in a contaminate using a chemical oxidation process.The treatment is effective for remediation of water-insoluble, toxic,carcinogenic and environmentally persistent organic compounds,particularly those with a bioavailability too low for effective use ofdirect bioremediation methods.

The chemical oxidation treatment of the present invention is mediated bya reactive free radical, that may be an oxidizing free radical producedby the action of a transition metal ion on an oxidizing agent at a pH inthe neutral range. The oxidizing free radical may be any oxidizing freeradical produced by the action of a transition metal ion on an oxidizingagent, such as for example the oxidizing free radicals .OH or .OOH,produced by the action of a transition metal ion:chelator complex onhydrogen peroxide.

The transition metal ion is stabilized by a metal chelator and the pH ismaintained by the addition of a buffering salt. The reactive freeradical initiates a chemical reaction with the organic compoundcontaminants that results in remediation of the contamination, such thatthe organic compounds are remediated. Preferably, remediation of theorganic contaminants results in one or more of the following: theorganic compound is solubilized, rendered more bioavailable, oxidized,degraded, decomposed, detoxified or mineralized.

In another aspect of the invention there is provided a method whichcombines oxidation and bioremediation of contaminants and which providesa means to control the pH to provide an environment suitable forchemical and biological transformations by microorganisms. Suitablemicroorganisms include any microorganism capable of oxidation of acontaminant, especially such microorganisms as bacteria, actinomycetesand fungi. A suitable microorganism may be used as a single microbialspecies. Preferably, however, the microorganisms exist as a populationof two or more species, which may be of different genera. Such apopulation of two or more suitable microorganisms is a herein referredto as a consortium. Yet more preferably, the consortium performs itsnatural degradative processes on the contaminants.

In yet another aspect the invention provides a method fordecontaminating or remediating contaminated soil and water by providingto a soil a consortium of microorganisms, selected to degrade knowncontaminants which may be present in the soil, and allowing themicroorganisms to degrade the contaminants in situ. Additionally, thesoil remediation provided by the selected consortium may be furtherenhanced by introducing chemical oxidation treatment, prior to,simultaneously with or subsequent to the introduction of the microbialconsortium. The oxidation is desirably carried out while maintaining abiocompatible pH to permit the consortium to degrade the contaminants.Preferably, the consortium is capable of this transformation as anatural degradation process, though selection may be applied to theconsortium to enhance the degradative activity.

A particular advantage of the methods taught herein is that thesemethods are compatible with the biological degradative processesmediated by the microorganisms introduced as microbial consortia, thoughthe introduction of single species of microorganisms to perform thisfunction is also contemplated. The degradative processes mediated by themicroorganisms or consortia may be natural processes of themicroorganisms.

Microorganisms present in a preferred consortium useful in the methodsof the present invention include Sphingomonas yanoikuyae, Sphingomonasspecies., and Pseudomonas species. In another preferred consortium themicroorganisms include Burkholderia species, Ochrobactrum species, andActinomyces species.

Other useful microorganisms in the methods of the present invention thatinvolve bioremediation include: Pseudomonas aeruginosa (ATCC 15522-28,21472); Pseudomonas species (NRRL 18064); Alcaligenes faecalis (ATCC8750); Alcaligenes eutrophus (NRRL 15940); Rhodotorula rubra (ATCC16639); Rhodococcus globerulus (NRRL 55255), and Xanthomonas maltophilia(ATCC 25556).

The microorganisms may be delivered by any one of the many well knownmethods. Examples include for instance, those described in the followingreferences which are not to be construed as limiting in any way:

Newcombe, D. A., and D. E. Crowley. 1999. Bioremediation ofatrazine-contaminated soil by repeated applications ofatrazine-degrading bacteria. Appl Microbiol Biotechnol. 51(6):877-82.

Barbeau, C, L. Deschenes, D. Karamanev, Y. Comeau, and R. Samson. 1997.Bioremediation of pentachlorophenol-contaminated soil by bioaugmentationusing activated soil. Appl Microbiol Biotechnol. 48(6):745-52.

The inventors have discovered a method of oxidation of hydrocarboncontaminants in situ that is biocompatible. This biocompatibility meansthat microorganisms may be employed at any of the various stages of themethod. This method therefore allows combined chemical oxidationtreatments with biodegradation steps with a microbiological culture orconsortium. The microbiological treatments steps bring the advantages ofmicrobiological remediation to the treatment methods of the presentinvention. These advantages include low environmental impact, low costof the microbiological culture or consortium, mild conditions of use andease of application of the microbiological culture or consortium to thecontaminate before, during or after the chemical remediation treatmentsteps as taught herein.

The inventors further provide a generally useful method of producing areactive free radical under very mild conditions in an aqueous medium ata neutral pH. The method essentially includes providing a transitionmetal in aqueous soluble form, along with a chelator of the transitionmetal, to form a transition metal:chelator complex; a buffering salt maybe included to maintain the pH in a neutral range and an oxidizing agentis added to provide the reactive free radical in the presence of thetransition metal:chelator complex. Such complexes are interchangeablyreferred to as transition metal:chelator complexes or chelates in thisspecification.

The standard Fenton's reaction involves the production of a hydroxylradical by the action of ferrous ions on hydrogen peroxide, to release aferric ion and a hydroxyl ion along with the hydroxyl radical. Theferric ion then catalyses the production of an oxyhydroxyl radical and ahydrogen ion from the hydrogen peroxide and reforms the ferrous ion.This reaction is carried out at low pH. The hydrogen peroxide isintroduced slowly to avoid explosive decomposition to oxygen catalyzedby the transition metal, which is in this case, iron.

In the methods of the present invention, a buffering salt is added tomaintain the acidity at a neutral or near neutral pH. The transitionmetal is further stabilized by the binding of the chelating agent. Thesemodifications prevents the precipitation of the transition metal as aninsoluble salt and further renders the entire process morebiocompatible. The bioavalable degradation products may then be furtherdegraded by any added microbial culture or consortium that isintroduced.

Addition of reagents may be in any order and as transition metalchelates or separately such that the chelates form in situ. In the casewhere the transition metal salt is a ferrous or ferric salt, thechelating agent stabilizes the ferric form and prevents precipitation ofthe ferric salts particularly ferric hydroxide.

Reaction times vary with the prevailing conditions, such as temperature,contaminant organic compound and reagent (transition metal ion,chelating agent and hydrogen peroxide) concentrations. Significantdegradation may occur over several days, though reaction times of lessthan 1-2 days are preferred and reaction times of 24 hours or less areoptimally preferred.

Delivery of the chemical reagents for the method of the presentinvention may be achieved by any of a variety of methods well known andset forth in the art. These include for example the methods described inthe following references:

Watts, R. J., D. R. Haller, A. P. Jones, and A. L. Teel. 2000. Afoundation for the risk-based treatment of gasoline-contaminated soilsusing modified Fenton's reactions. J Hazard Mater. 76(1):73-89.

Kao, C. M., and M. J. Wu. 2000. Enhanced TCDD degradation by Fenton'sreagent preoxidation. J Hazard Mater. 74(3):197-211.

Arienzo. M. 2000. Use of abiotic oxidative-reductive technologies forremediation of munition contaminated soil in a bioslurry reactor.Chemosphere. 40(4):441-8.

Hayes, T. D., D. G. Linz, D. V. Nakles, and A. P. Leuschner (Eds.).1996. Management of manufactured gas plant sites, vol. 2. [p. 427-437:Chemical Oxidation]. Amherst Scientific Publishers, Amherst, Mass.

The combination of chemical oxidation and biodegradation of the presentinvention has the great advantage over either treatment alone in theremediation of organic contaminants: The use of Fenton's reagent withbiodegradation provides increased effectiveness in part because oxidizedorganic compounds (such as polyaromatic hydrocarbons, PAHs) are morewater soluble than the original contaminants. The increased solubilityleads to higher bioavailability and in turn the higher bioavailabilityresults in more extensive remediation.

The inventors have discovered that oxidation methods for remediation ofcontaminates using transition metals and oxidizing agents accompanied bythe introduction of a complexing or chelating agent for the transitionmetal can be conducted without the usual decrease in soil pH. Thisfeature of pH maintenance provides several advantages. For example, theuse of the complexing or chelating agent to modify Fenton's reagentchemistry allows for the maintenance of pH at levels suitable to sustainbacteria and further permits microbial action to enhance the remediationprocess.

The advantages of maintaining biocompatible pH levels include theability to contemporaneously obtain effective bioremediation bynaturally occurring and/or introduced bacteria. Furthermore, use of achelating agent eliminates the concern of an acidified soil environmentwhich is associated with the original Fenton reaction. Among suchcomplexing agents are included hydroxylated benzenes, which have beenfound to be especially useful. More desirably, dihydroxybenzene providesparticularly desirable results.

The transition metals useful in practicing the present invention includemanganese, iron, cobalt, nickel, copper and zinc, with iron salts beingpreferred, the iron salt preferably being a sulfate, a perhalate or anitrate. More preferably the iron is a Fe(II) ferrous salt or a ferricFe(III) salt, and most preferably as an Fe(III) ionic species. TheFe(III) may be a sulfate or a nitrate, but is preferably a perhalate,such as perchlorate, perbromate or periodate, with perchlorate beingoptimally preferred.

Oxidizing agents useful in the methods of the present invention may beany of a number of oxidizing agents that provide a reactive free radicalin the presence of a transition metal:chelator complex at a pH in theneutral range. Among the useful oxidizing agents are peroxides such asfor example, hydrogen peroxide, which is especially preferred.

The chelating agents useful in the present invention may be anychelating agent that forms a transition metal chelate with thetransition metal chosen for production of the reactive oxidizing freeradical. Hydroxylated aromatic compounds are especially useful aschelating agents in these methods. Examples of useful chelating agentsinclude hydroquinone, orcinol, resorcinol, trihydroxybenzene,salicylate, m-hydroxybenzoate, p-hydroxybenzoate, nitrilotriacetic acidand diethylenetriaminepentaacetate. Preferred chelating agents for usein combination with iron as Fe(II) or Fe(III) as the transition metalinclude the hydroxybenzenes and the hydroxybenzoic acids. Optimally, thechelating agents for use in combination with Fe(II) or Fe(III) arecatechol (1,2 dihydroxybenzene) or gallic acid (3,4,5 trihydroxybenzoicacid).

The chelators are generally used in amounts sufficient to chelate theamount of transition metal introduced into the contaminate system.Desirably the ratio of transition metal:chelator complex to oxidizingagent may be any ratio from about five parts peroxide to one part ironby weight to about twenty parts peroxide to one part iron by weight.Optimally, the ratio of peroxide to iron is about ten parts peroxide toone part iron by weight.

It is important to note that the optimal ratio of peroxide to iron byweight may be affected by the type of contaminate and the amounts andthe chemical composition of the contaminants therein. This ratio may beroutinely determined for each contaminate on a small scale and appliedon the a larger scale to the remediation or decontamination project athand.

Gallic acid is among the hydroxybenzene chelating agents especiallyuseful in the methods of the present invention. Gallic acid may beproduced from plant tannins by standard well known chemical processes.The Materia Medica gives the following list of plant genera (along withexamples given their common names) that are high in tannins and whichwould therefore be of value as inexpensive sources of tannins for theproduction of hydroxybenzene chelating agents, particularly gallic acid:Abies (Spruce), Agrimonia (Agrimony), Alnus (Alder), Betula (Birch),Cinnamomum (Cinnamon), Cola nitida (Cola Nuts), Ephedra (Mormon Tea),Fraxinus (Ash), Geranium (Cranesbill, Alum Root), Granatum (Punica,Pomegranate), Guaiacum (Lignum Vitae), Hamamelis (Witch Hazel), Heuchera(American Alum Root), Juglans (Walnut, Butternut), Ligustrum (Privet),Myrica (Bayberry), Orobanche (Broomrape), Potentilla (all), Prunus (Wildor Choke Cherry), Quercus (Oak), Rheum (Rhubarb), Rhus (all: Sumach),Rosa (Rose), Rubus (Blackberry, Raspberry), Vaccinium(Blue-/Huckle-/Bilberry), Xanthium (Cocklebur).

Without wishing to be bound by any particular theory, it is believedthat the presence of the chelating agent stabilizes the reactivetransition metal ion in the complex and prevents precipitation as aferric salt without inhibiting the catalytic activity of the transitionmetal ion. The transition metal ion in the complex interacts with theoxidizing agent in the reaction mixture enhancing the production of ahighly reactive oxidizing free radical. The avoidance of the low pHprevents solubilization of heavy metals and enhances thebiocompatibility of the reaction. The products of the remediationreaction formed when contaminates react with the highly reactiveoxidizing free radical are also generally less toxic than the productsof the commonly available harsher chemical remediation methods.

In addition to chemical treatment, the present invention providescombined chemical remediation and bioremediation techniques. Althoughnaturally occurring microorganisms may be relied upon to performdegradative processes contemporaneously with the chemical treatmentprocess of the present invention, it is desirable to select a consortiumof microorganisms known to be effective at degrading the contaminants inthe soil, and introducing such a consortium prior to, during orsubsequent to such chemical treatment.

For example, a consortium known to be effective in degrading PAHs suchas, a consortium of Burkholderia spp., Ochrobactrum spp., andActinomyces spp. can be introduced into the soil, and permitted tocontemporaneously work along with chemical oxidation treatment. In thisway, the advantages of both chemical treatment and bioremediation can beobtained.

The invention also provides a kit for treatment of a contaminatecontaminated with an organic compound, the kit includes: a transitionmetal in soluble form; a chelator of the transition metal that has theproperty of forming a transition metal:chelator complex with thetransition metal; and a buffering salt that is soluble in acidicsolutions. The buffering salt may have a pK_(a) suitable to maintain thepH in the neutral range. Preferably the buffering salt has a pK_(a) inthe range from about 5 to about 8.

The transition metals and transition metal chelators useful in the kitsof the present invention are as described above. The transition metalchelator is most preferably catechol or gallic acid. The kits preferablyalso contain an oxidizing agent that reacts with the transitionmetal:chelator complex to form a reactive free radical. The preferredoxidizing agent is hydrogen peroxide.

The kit may further comprise a microbial consortium having the propertyof solubilizing or biodegrading an organic compound contaminant.Preferably the microbial consortium comprises one or more of thefollowing: a bacterial species, a fungal species and an actinomycesspecies. The bacterial species, a fungal species and an actinomycesspecies that are particularly useful in the kits of the invention havebeen described above. These include one or more of the following: anAlcaligenes species, a Sphingomonas species, a Pseudomonas species, aRhodotorula species, a Burkholderia species, an Ochrobactrum species, aRhodococcus species, a Xanthomonas species and an Actinomyces species.

The following examples were conducted to test the efficacy and sequenceof combined chemical oxidation and biodegradation in the remediation ofsoils contaminated with a mixture of PAH compounds from the site of aformer manufactured gas plant (MGP).

EXAMPLES

Soil Samples and Chemicals. Quakertown silt loam was collected from thedepth of 0 cm to 15 cm below the surface at the Snyder farm of RutgersUniversity (Pittstown, N.J.). The soil is free of PAHs and has neverbeen exposed to anthropogenic input of PAH compounds. The soil wasair-dried, passed through a 2-mm sieve, and sterilized by gammairradiation (2.5 Mrad) from a ⁶⁰Co source (Ward laboratory, CornellUniversity, Ithaca, N.Y.). The soil consisted of 36% sand, 54% silt, and10% clay. It contained 2.94% organic carbon and had a pH of 5.9 in water(1:1). Coal tar-contaminated soil was collected from a formermanufactured gas plant site in New Jersey. The soil was classified asloamy sand, consisting of 78% sand, 11% silt, and 11% clay.

Seven PAHs, including naphthalene (NAP), fluorene (FLU), phenanthrene(PHE), anthracene (ANT), pyrene (PYR), chrysene (CHR), benzo(a)pyrene(BaP), and also radiolabeled [7-¹⁴C]BaP (specific activity, 26.6mCi/mmol; purity, >95%) were used for this study. All chemicals werepurchased from Sigma Chemical Company. Ten milliliters of stock solutionwere made in a 20-mL amber vial with a Teflon-lined cap by dissolving 10mg each of NAP, FLU, PHE, ANT, and PYR and 5 mg each of CHR and BaP in 1mL of dichloromethane. For artificial contamination of PAHs, 10 g ofQuakertown silt loam were placed in a 125-mL flask (or a 100-mL glassbottle) and spiked with 100 μL of the stock solution. The solvent wasallowed to evaporate in a fume hood for an hour with mixing the soil in15-min intervals. This gave a total PAHs concentration of 600 μg pergram of soil. The physical and chemical properties of the PAHs used forthis study are presented in Table 1.

TABLE 1 Physical and Chemical Properties^(a) of PAHs Used andConcentrations of PAHs in a Former MGP Soil Solubility Vapor RelativeNumber in water Pressure Initial conc. Abundance PAHs of Rings(mg/L)^(b) Log K_(ow) ^(c) (at 20° C.) (mg/Kg soil)^(d) (%) Naphthalene2 32 3.37 4.9 × 10⁻² 1205 (101) 32.4 fluorene 3 1.9 4.18 1.3 × 10⁻² 252(31) 6.8 phenanthrene 3 1.0 4.46 6.8 × 10⁻⁴ 921 (89) 24.7 anthracene 30.07 4.45 1.9 × 10⁻⁴ na^(e) na pyrene 4 0.16 5.32 6.8 × 10⁻⁷ 524 (36)14.1 chrysene 4 0.006 5.61 6.3 × 10⁻⁷ 454 (62) 12.2 benzo(a)pyrene 50.0038 6.04 5.0 × 10⁻⁷ 366 (59) 9.8 ^(a)Data from Sims, R. C. andOvercash, M. R. Residues Rev (1983) 88: 1-68; and Lee, L. S., Roa, P. S.C. and Okuda, I. Environ. Sci. technol. (1992) 26: 2110-2115.^(b)Crystal solubility at 25° C. ^(c)Logarithm of the octanol: waterpartition coefficient. ^(d)Values are the means of nine replicatedeterminations (standard deviations). ^(e)Not assayable.

The microbial Consortium utilized in the following examples was culturedfrom coal tar-contaminated soil. The following enrichment culturetechnique was used to isolate PAH-degrading organisms as a consortium:Two- to five-gram samples of MGP soil collected from the depth of 0-2 mbelow surface were incubated with a mixture of PAHs in 100 mL ofinorganic salts solution (0.10 g CaCl₂.2H₂O, 0.01 g FeCl₃, 0.10 gMgSO₄.7H₂O, 0.10 g NH₄NO₃, 0.20 g KH₂PO₄, and 0.80 g K₂HPO₄/L of dH₂O;pH 7.0) at 30° C. for two weeks. PAHs including PHE, ANT, PYR, CHR, andBaP were dissolved in methanol (10 mg/mL for the first three compoundsand 1 mg/mL for the others), and the PAHs-methanol solution was used assubstrates for the enrichment. After two weeks of incubation, 10 mL ofthe supernatant were collected and incubated for two more weeks asdescribed above. By this procedure, a consortium capable of degrading avariety of PAHs was obtained and used for biodegradation experiments.The consortium was maintained in 50 mL of inorganic salts solutioncontaining 50 mg of phenanthrene as a sole carbon source. After 5 daysof incubation at 30° C. with shaking (200 rpm), the culture wascentrifuged at 7600×g for 10 min. The cells were washed twice with theinorganic salts solution and an inoculum of more than 10⁸ cells was usedfor biodegradation. The number of viable cells was determined by platecounting on Trypticase Soy Agar (TSA).

In this way, the particular species in the consortium that flourished inthe medium were represented by a much higher population number after twoweeks of growth on the contaminant mixture expected in the particularapplication. This mixture was thereafter saved and frozen for furtheruse later.

A transition metal is needed to react with the peroxide to free areactive free radical. The transition metals include manganese, iron,cobalt, nickel, copper and zinc. Iron is the preferred transition metalfor practicing the present invention. Of particular interest is FeSO₄,or Fenton's reagent.

Fenton's reagent was generated by mixing hydrogen peroxide (30%, w/v)with FeSO₄.7H₂O in varying ratios to determine the optimum conditionsfor the degradation of PAHs. The sequence of reagent addition seemed tobe important to ensure that the reaction occurred efficiently, as wellas to ensure that the reaction could be safely maintained. Desiredamounts of ferrous sulfate were added to 10 g of PAHs-spiked soil or 5 gof MGP soil in 20 mL of distilled water containing 0.02% HgCl₂. To thesoil slurry, hydrogen peroxide was introduced gradually by pipetting(ca. 0.3 g each with 10-min intervals), and the reaction was allowed tooccur for 24 h on an orbital platform shaker at room temperature.Preliminary experiments showed that stepwise addition of hydrogenperoxide was more effective compared to bulk-type addition in batches.For the Fenton-type reaction at near neutral pH, a modified Fenton'sreagent was developed by using a chelating agent (i.e., catechol orgallic acid) and Fe(ClO₄)₃.6H₂O. Two grams of hydrogen peroxide and 323mg of Fe(ClO₄)₃.6H₂O (0.07 M) were used for the reaction. The molarconcentration of chelating agent was the same as that of Fe³⁺; 82.5 mgof catechol or 141.0 mg of gallic acid was used. Before the addition offerric ions and hydrogen peroxide, an appropriate chelator was added tothe soil slurry and 1 g of calcium carbonate was also applied tomaintain the pH of the system at about 6.0 to 6.5 throughout thereaction.

When Fenton-type reaction was combined with biodegradation, inorganicsalts solution (pH 7.0) instead of distilled water was used to promotemicrobial degradation. An active microbial consortium described abovewas used for biodegradation. Biodegradation was performed for four weeksat room temperature on a rotary shaker.

Extraction and Determination of PAHs. The soil slurry used for eitherbiodegradation or Fenton-type reaction, or both, was transferred to a50-mL Teflon centrifuge tube and centrifuged at 18,600×g for 15 min.After removing the supernatant, 10 mL each of dichloromethane andacetone were added to the soil and the soil-solvent suspension wasshaken (200 rpm) for 48 h at 30° C. for the extraction of PAHs. The tubewas then centrifuged at 18,600×g for 15 min and the solvent mixture wastransferred to a 50-mL test tube. After removing excess water (upperlayer; ca. 2 mL) by pipetting, 4 g of anhydrous sodium sulfate weremixed with the PAHs-containing solvent to remove residual watercompletely from solvent. The concentration of PAHs in the water layerwas less than the detection limit of the analytical procedure used inthis study. The extract was then concentrated to 1 to 2 mL using anevaporator (Büchi Rotavapor; Buchler Instruments Inc., Fort Lee, N.J.)for further analysis. By this procedure, 100% of the PAHs freshly spikedto Quakertown silt loam were recovered.

The extract was passed through a 0.45-μm PTFE syringe filter to removeany particulates present and analyzed by a gas chromatograph (GC)equipped with a flame ionization detector (Varian Star 3500; VarianChromatograph Systems, Walnut, Calif.). The GC was installed with aRtx-5 silica column crossbonded with 5% diphenyl and 95%dimethylpolysiloxane (30 m×0.53 mm inner diameter; Restek Corporation,Bellefonte, Pa.). The oven temperature was programmed at 40° C. for 6min, followed by a linear increase of 10° C. per minute to 300° C., andthen the temperature was held for 15 min. Injector and detectortemperatures were maintained at 300° C. Two microliters of the extractwere injected and nitrogen was used as a carrier gas.

Data Analysis. Precautions were taken to correct for differences inextractability and volatilization of PAHs that occurred during thefour-week biodegradation experiment. Since the extractability of organiccompounds decreases as the residence of the compounds in soil increases(15) we have determined the time dependency of the efficiency of theextraction method used for this study with PAHs-spiked soil samples. Adecreased extractability was observed, especially with CHR and BaP. Tocorrect for this, separate sets of soil samples spiked with PAHs weremade in parallel with soil samples for treatments, and run as controlsduring the incubation period of each treatment. In MGP soils, it wasassumed that extractability of PAHs did not decrease during the periodof treatment because the PAHs in the soils had been present for over 100years. Instead, concentrations of PAHs were not uniform in MGP soils,which would influence the efficiency of chemical and biologicaltreatments. Thus, the soil samples were mixed thoroughly each time whenthey were used, and separate sets of samples were also run as control.Values presented in the tables represent the percentages of the amountsof PAHs recovered from each control of corresponding incubation period.

Comparative Example 1

Biodegradation of PAHs by a Microbial Consortium. The ability of ahighly active consortium that had been obtained from the MGP to degradePAHs was tested by spiking a mixture of PAHs into a model soil. The PAHsranged from two- to five-ring compounds and were freshly spiked toQuakertown silt loam for biodegradation. As shown in Table 2, thisconsortium immediately degraded two-ring compounds and PHE. More than94% of ANT also disappeared in two weeks of biodegradation. PYR and CHRshowed slower degradation, but eventually more than 95% were degraded.Noticeably, this consortium was capable of degrading more than 70% ofBenzo(a)pyrene in the model soil.

TABLE 2 Biodegradation of Polyacyclic Aromatic Hydrocarbons (PAHs)Freshly Added to Quakertown Silt Loam by a Microbial Consortium % ofcontrol remaining in soil^(a) PAHS 1 week 2 weeks 3 weeks 4 weeksNaphthalene nd^(b) Nd nd nd Fluorene 4.6a 2.2b 1.7b 1.5b Phenanthrene Nd2.7a 1.6ab 1.2b Anthracene 23.3a 5.5b 3.4bc 2.5c Pyrene 62.4a 9.9b 5.6c4.8c Chrysene 97.7a 28.2b 10.2c 7.4c Benzo(a)pyrene 94.2a 59.5b 31.6c27.4c ^(a)Values are the means of triplicate determinations. Values in arow followed by the same letter are not significantly different (p <0.05). ^(b)Not detected.

Comparative Example 2

Fenton's Reagent Treatment. To obtain an optimum condition for Fentonreaction in the transformation of freshly added PAHs, hydrogen peroxideoxidation was performed in the presence of ferrous sulfate with varyingratios of peroxide to ferrous ions in the model soil (Table 3). At theratio of 1:1 ([H₂O₂:FeSO₄]=[0.2 g:0.2 g]), oxidation of PAHs was notefficient except for NAP, which was reduced to 35% of the initialamount. Increasing the amount of hydrogen peroxide enhanced theoxidation efficiency. At the ratio of 10:1 ([H₂O₂:FeSO₄]=[2.0 g:0.2 g]),a large amount of the PAHs was destroyed by a 24-h oxidation. Amounts ofNAP, FLU, and PHE were reduced by 90 to 100% and PYR, a four-ringcompound, was also readily destroyed. However, ANT and CHR showed somedegree of resistance to the hydrogen peroxide treatment since only about40 and 12%, respectively, was degraded by the same treatment.Unexpectedly, the very recalcitrant hydrocarbon Benzo(a)pyrene was oneof the most sensitive hydrocarbons to the oxidation and only 3.3%remained in the model soil.

TABLE 3 Effect of Fenton Reaction on the Degradation of PAHs by VaryingRatios of Hydrogen Peroxide to Ferrous Sulfate in Quakertown SiltLoam^(a) % remaining in soil PAHs 1.1^(b) 5:1^(c) 10:1^(d) 10:1^(e)Naphthalene 35.2a nd^(f) <9.3b^(g) nd Fluorene 76.5a 44.5b 11.0c 18.8dPhenanthrene 83.7a 48.2b nd nd Anthracene 100a 75.9b 61.9c 79.3b Pyrene95.4a 51.0b 15.5c 26.3d Chrysene 100a 87.0b 88.1b 98.2c Benzo(a)pyrene84.8a 35.3b 3.3c 60.9d ^(a)Values are the means of five replicatedeterminations. Values in a row followed by the same letter are notsignificantly different (<0.05). ^(b)[H₂O₂:FeSO₄] = [0.2 g:0.2 g]^(c)[H₂O₂:FeSO₄] = [1.0 g:0.2 g] ^(d)[H₂O₂:FeSO₄] = [2.0 g:0.2 g]^(e)[H₂O₂:FeSO₄] = [1.0 g:0.1 g] ^(f)Not detected. ^(g)Detected fromonly one sample.

Since the ratio of 10:1 was shown to be the most effective in thedegradation of PAHs, the amounts of hydrogen peroxide and ferrous ionswere changed while maintaining the same ratio. When 1 g of hydrogenperoxide and 100 mg of ferrous sulfate were used, the degradationefficiency for NAP, FLU, and PHE was not significantly altered comparedto when 2 g and 200 mg of each reagent was used. However, PAHs includingANT, PYR, CHR, and BaP were degraded to a significantly lesser extent(Table 3). In one experiment, the reaction (2 g H₂O₂ and 200 mg FeSO₄)was extended to 3 days to see if further degradation was evident, but noadditional benefit was obtained.

The amount of ferrous sulfate was changed while maintaining the amountof hydrogen peroxide constant (2 g). As shown in Table 4, the efficiencyof PAHs degradation greatly decreased when ferrous ions were not addedto the reaction. Even with the most susceptible hydrocarbon NAP, morethan 56% of the initial amount remained in the model soil. When 100 mgof ferrous sulfate was added, the efficiency was almost the same as withthe reaction with 200 mg of ferrous sulfate except for ANT and BaP.Degradation of hydrocarbons was significantly enhanced by the additionof 200 mg of ferrous sulfate.

TABLE 4 Effect of Fenton Reaction on the Degradation of PAHs in thePresence of Hydrogen Peroxide (2g) and Various Amounts of FerrousSulfate in Quakertown Silt Loam^(a) % remaining in soil PAHs No FeSO₄100 mg 200 mg^(b) Naphthalene 56.1a nd^(c) <9.3b Fluorene 88.7a 19.8b11.0c Phenanthrene 88.3 Nd nd Anthracene 92.7a 76.3b 61.9c Pyrene 88.3a16.3b 15.5c Chrysene 77.0ab 72.6a 88.1b benzo(a)pyrene 73.2a 10.5b 3.3c^(a)Values are the means of five replicate determinations. Values in arow followed by the same letter are not significantly different (p <0.05). ^(b)Data from TABLE 3. ^(c)Not detected.

Comparative Example 3

Biodegradation vs. Fenton Reaction in MGP Soil. The biodegradingactivity of the consortium was also determined in an MGP soil that washeavily contaminated with PAHs. Initial concentrations of PAHs weredetermined by using dichloromethane and acetone as extraction solvents(Table 1). The soil contained 63.9% of easily biodegradable PAHs (i.e.,NAP, FLU, and PHE) and 36.1% of four- and five-ring hydrocarbons. A peakcorresponding to ANT was not clearly separable and sometimes overlappedwith a PHE peak on the GC chromatogram, hence ANT was not included inthis analysis.

Fenton reaction in the ratio of 10:1 ([H₂O₂:FeSO₄]=[2.0 g:0.2 g]) wasused to investigate degradation of PAHs in MGP soil. For NAP, FLU, andPHE, 80 to 99% of the initial amount was removed, but only 20 to 40% wasdegraded for higher molecular weight PAHs such as PYR, CHR, and BaP(Table 5). The degradation efficiency of PAHs was even higher bybiodegradation. NAP disappeared completely during the four-weekbiodegradation treatment and about 90 and 84% of FLU and PHE weredegraded, respectively. Amounts of four- and five-ring hydrocarbons werereduced by about 35 to 50% (Table 5). When compared to the model soil,PAHs degradation by either biodegradation or Fenton reaction was not asefficient in MGP soil, especially for PYR, CHR, and BaP, probably due toheavy contamination of the soil.

TABLE 5 Efficacy of Fenton Reaction and Biodegradation in the Removal ofPAHs in MGP Soil % remaining in soil^(a) PAHs Bio^(b) FR^(c) Naphthalene<1.11 nd Fluorene 15.6a 10.3a Phenanthrene 20.0a 16.8a Pyrene 59.8a53.4a Chrysene 77.2a 50.8b benzo(a)pyrene 79.5 64.9  ^(a)Values are themeans of five replicate determinations. Values in a row followed by thesame letter are not significantly different (p < 0.05).^(b)Biodegradation for four weeks at room temperature. ^(c)Fenton'sreagent treatment for 24 hours.

Fate of Benzo(a)pyrene during Fenton Reaction. It is theoreticallypossible that Fenton's reagent could completely transform organiccompounds to carbon dioxide (“mineralization”). However, little attempthas been made to quantify how much of the parent compounds aremineralized in a typical Fenton reaction. For this test, Benzo(a)pyrenewas chosen as a model hydrocarbon, and the distribution ofBenzo(a)pyrene during the Fenton reaction was monitored by using¹⁴C-labeled compound and gas chromatography (Table 6). Results showedthat only 3.3% of the initial amount was recovered as the parentcompound. Of the remaining, 18.3% was detected as carbon dioxide, and anadditional 16.8 and 31.3% was detected as intermediates from aqueousphase and soil extract, respectively. The remaining 33.6% of the initialBenzo(a)pyrene was not extractable by the solvent extraction used forthis study. This portion may represent either oxidized intermediatescovalently bound to soil organic matter or parent compounds sequesteredin the soil matrix, or both.

TABLE 6 Fate of Benzo(a)pyrene During the Treatment of Fenton's Reagentin Quakertown Silt Loam^(a) % recovered parent compound^(b) 3.3Mineralization to CO₂ 18.3 Intermediates Water soluble 16.8 CH₂Cl₂extractable 31.3 Nonextractable (calculated)^(c) 30.3 Total 100^(a)[H₂O₂:FeSO₄] = [2.0 g:0.2 g] ^(b)Determined by GC/FID ^(c)Thisfraction may include intermediates incorporated into soil organic matterand parent compounds strongly sorbed to soil.

In order to inhibit the reaction of the transition metal with theperoxide, maintain a moderate pH, and in order to solubilize thetransition metal, a weak complexer or chelator is utilized. Thecomplexer is desirably biodegradable. The complexer must be chosen so itis not too strong a chelator to prevent the transition metal toparticipate in free radical generation, i.e. it must be sufficientlyweak to release the metal for its function. The complexing agent weaklycoordinates with the transition metal to provide aqueous solubility.Preferably an hydroxylated benzene such as catechol or gallic acid isused. These compounds are naturally occurring in soil and do not add tothe contamination, nor detract from the biocompatibility of thetreatment.

Example 5

Modified Fenton's Reagent. In an attempt to remove more PAHs from theMGP soil, biodegradation in conjunction with Fenton-type oxidation wouldappear to be a viable option. However, the extremely low pH requirement(optimum pH ca. 2-3) for the Fenton reaction makes the processincompatible with biological treatment. In addition, this low pH mayincrease mobilization of heavy metal co-contaminants, and woulddevastate the soil ecosystem where the reagent is used. In order toovercome these limitations, two approaches were used. In the firstapproach, the pH of the system was adjusted to approximately 6 byaddition of alkali (i.e., NaOH), buffer solutions (i.e., potassiumphosphate buffer, phosphate-buffered saline), or calcium carbonate.However, this approach failed to produce the desired result. Either thepH of the system dropped to pH 2 (for alkali and buffer solutions) orthe efficiency of the reaction decreased greatly (for calcium carbonate;second column in Table 7). As a second approach, the Fenton's chemistrywas modified by the use of chelating agents such as catechol or gallicacid, and by the use of ferric ions instead of ferrous ions. Calciumcarbonate was used to maintain the pH of the system around 6 to 6.5throughout the reaction. Fifteen potential chelators including di- andtrihydroxybenzenes, di- and trihydroxybenzoic acids, gallic acid,nitrilotriacetic acid, diethylenetriaminepentaacetate, and salicylicacid were tested to identify an appropriate chelator. Among them,catechol and gallic acid were chosen based on their performance withregard to the degradation efficiency of PAHs. Destruction efficiency wassimilar between catechol and gallic acid and use of this modifiedFenton's reagent allowed for destruction of PAHs (Table 7). As found forthe unmodified Fenton reaction, PYR and BaP were more sensitive to thehydrogen peroxide oxidation than ANT and CHR. On the whole, the modifiedFenton's reagent resulted in a decline in overall performance relativeto the unmodified Fenton's reagent, but the pH was maintained at about 6to 6.5, which allowed for the combined treatment with biodegradation(Table 7).

TABLE 7 Degration of PAHs by Hydrogen Peroxide Oxidation at Near NeutralpH (ca. 6.0-6.5) in Quakertown Silt Loam % remaining in soil^(a)FR^(b) + mFR^(c) PAHs CaCO₃ + catechol + gallic acid Naphthalene 16.1a15.3a 11.1a Fluorene 72.5a 67.2ab 63.8b Phenanthrene 82.0a 68.3b 70.1bAnthracene 78.1a 70.8ab 66.8b Pyrene 77.1a 58.3b 58.9b Chrysene 100a100a 91.1b banezo(a)pyrene 73.1a 54.8b 49.6b ^(a)Values in columns arethe means of five replicate determinations. Values in a row followed bythe same letter are not significantly different (p < 0.05). ^(b)FRstands for Fenton's reagent. ^(c)mFR stands for modified Fenton'sreagent.

Example 6

Biodegradation Combined with Modified Fenton's Reagent. The modifiedFenton's reagent has been tested in combination with biologicaltreatment using the PAH-degrading microbial consortium described above.Two possible treatment sequences were tested: modified Fenton's reagentfollowed by biodegradation and biodegradation followed by Fenton'streatment. The results in Table 8 show that the sequence in which thecombined treatment was carried out had a pronounced effect on theoutcome. Biodegradation followed by modified Fenton's treatment wassuperior to the reverse-order sequence in the degradation of PAHs in MGPsoil. In both sequences, NAP was degraded almost completely. Whenmodified Fenton's reagent was followed by biodegradation, about 31 to39% of FLU and PHE were recovered from the MGP soil and about 62 to 86%of PYR, CHR, and BaP remained in the soil. However, between 85 and 98%of initial NAP, FLU, PHE and PYR were removed from the soil by thereverse-order sequence, using catechol as the ligand (Table 8). Ingeneral, catechol was a slightly better chelator than gallic acid. Theresults show that there are distinct advantages to using a modified formof Fenton's reagent in combination with biodegradation for theremediation of PAHs-contaminated soil.

TABLE 8 Combined Effect of Modified Fenton's Reagent and Biodegradationon the Degradation of Aged PAHs in a MGP Soil % remaining in soil aftertreatment^(a) MFR→Bio^(b) Bio→mFR PAHs Catechol gallic acid catecholgallic acid Naphthalene 5.88a 5.67a nd^(c) nd Fluorene 31.4a 34.5a 1.37b2.53b Phenanthrene 33.1a 38.9a 1.23b 1.39b Pyrene 62.2a 74.8b 14.2c24.6d Chrysene 85.9a 81.7a 34.2b 48.7c banezo(a)pyrene 74.1a 75.5a 32.3b44.1c ^(a)Values are the means of five replicate determinations. Valuesin a row followed by the same letter are not significantly different (p< 0.05). ^(b)mFR: modified Fenton's reagent and biodegradation wasperformed for four weeks at room temperature. ^(c)Not detected.

The original Fenton's reagent (H₂O₂+FeSO₄) reduced significant amountsof total PAHs, and especially BaP, in a model soil. However, efficiencydeclined greatly when used in an MGP soil. This might have resulted fromthe heavy contamination of PAHs, and co-contamination by other types ofhydrocarbons (e.g., aliphatics), in the MGP soil. By GC analysis, morethan 100 peaks were identified in solvent extracts of the MGP soil. Dueto the nonspecificity of hydroxyl radicals, it is likely that theradicals generated were scavenged by other organic compounds present inthe MGP soil as well as by the PAHs. In addition, natural organic mattercan be another sink for hydroxyl radicals in soil. Soil organic mattercan reduce the efficacy of Fenton-type reaction by competing withcontaminants for hydroxyl radicals or by catalyzing hydrogen peroxidedecomposition (Pignatello, J. J., Chapa, G. 1994. Degradation of PCBs byferric ion, hydrogen peroxide and UV light. Environ. Toxicol. Chem. 13(3), 423-327; Ronen, Z., Morrath-Gordon, M., Bollag, J. M. 1994.Biological and chemical mineralization of pyridine. Environ. Toxicol.Chem. 13 (1), 21-26). This suggests that results from simple systems(e.g., Fenton-type oxidation in organic-free water) may not be directlyuseful for predicting the degradation of contaminants present inmatrices that also contain natural organic matter.

The ratio of hydrogen peroxide to ferrous ions is known to be animportant determinant in the efficacy of the Fenton oxidation. Pratapand Lemley (Pratap, K., Lemley, A. T. 1998. Fenton electrochemicaltreatment ofaqueous atrazine and metolachlor. J. Agric. Food Chem. 46(8), 385-3291) reported that a ratio of 5:1 was the most efficient indegradation of atrazine and metolachlor while Arnold et al. (Arnold, S.M., Hickey, W. J., Harris, R. H. 1995. Degradation of atrazine byFenton's reagent: condition optimization and product quantification.Environ. Sci. Technol. 29 (8), 2083-2089) observed complete degradationof atrazine at a ratio of 1:1. In addition, Tyre et al. (Tyre, B. W.,Watts, R. J., Miller, G. C. 1991. Treatment of four biorefractorycontaminants in soils using catalyzed hydrogen peroxide. J. Environ.Qual. 20 (4), 832-838) observed that the highest degradation efficiencyof pentachlorophenol and dieldrin occurred without addition of exogenousiron, for soils that contained iron minerals. In our system, a 10:1ratio allowed for the greatest degradation of PAHs. The need for thehigher ratio in our system may be due to types and concentrations ofcontaminants, characteristics and content of soil organic matter, orsoil mineralogy.

Our results showed that biodegradation of low molecular weight PAHs wasfaster and more extensive than that of high molecular weighthydrocarbons. However, this is not consistent with the degradationpattern obtained using Fenton's reaction. Despite the general consensusthat hydroxyl radicals are nonspecific oxidants, PYR and BaP seemed tobe very susceptible and ANT and CHR were shown to be resistant toFenton-type oxidation in a model soil. BaP is known to be veryrecalcitrant to microbial degradation and thus persistent in theenvironment. In this regard, the finding that BaP is readily destroyedby hydroxyl radicals is noteworthy although the mechanism(s) by whichthus occurs can not be explained from our experiments. By using theFenton-type oxidation, it seems to be possible to bring about a nearlycomplete transformation of hydrocarbons that are only slightlybiodegradable such as Benzo(a)pyrene.

The fact that the optimum pH for a conventional Fenton reaction is pH 2to 3 poses great concerns ecologically. First, low pH itself can resultin significant environmental perturbation and second, low pH can enhancethe solubility of heavy metal ions that may be present asco-contaminants. The efficiency of the Fenton reaction greatly decreaseswith increasing pH (Arnold, S. M., Hickey, W. J., Harris, R. H. 1995.Degradation of atrazine by Fenton's reagent: condition optimization andproduct quantification. Environ. Sci. Technol. 29 (8), 2083-2089)because the solubility of ferric ions (converted from ferrous ions byhydroxyl radicals) declines at higher pH (i.e., above pH 3). The declinein reactivity is due to precipitation of ferric ions as an oxyhydroxidecomplex (Bohn, H. L., McNeal, B. L., O'Conner, G. A. 1985. In: SoilChemistry. John Wiley & Sons, New York. pp. 21-65). However, aFenton-type reaction can occur at near-neutral pH by stabilizing thesolubility of ferric ions with chelating agents (Sun, Y., Pignatello, J.J. 1992. Chemical treatment of pesticide wastes. Evaluation of Fe(III)chelates for catalytic hydrogen peroxide oxidation of 2,4-D atcircumneutral pH. J. Agric. Food Chem. 40 (2),322-327; Sun,Y.,Pignatello, J. J. 1993. Activation of hydrogen peroxide by iron (III)chelates for abiotic degradation of herbicides and insecticides inwater. J. Agric. Food Chem. 41 (2), 308-312; Pignatello, J. J., Baehr,K. 1994. Ferric complexes as catalysts for “Fenton” degradation of 2,4-Dand metolachlor in soil. J. Environ. Qual. 23 (2), 365-370). Among thechelators tested during this study, catechol and gallic acid resulted inthe most efficient degradation of PAHs in a model soil. Since bothcatechol and gallic acid are natural products and are readily degradedby microorganisms, their use as chelators is not likely to pose anenvironmental concern. It should be noted that the pH of the systemremained above 6 during the reaction using either catechol or gallicacid. Although this modified form of the Fenton reaction was not asefficient as the original version, the modification has the greatadvantage of being directly compatible with biodegradation processes.

The primary objective of the present study was to determine whether abiologically compatible Fenton oxidation, in combination withbiotreatment, could achieve maximum degradation of contaminants in soil.Integration of chemical oxidation with biodegradation can be performedin two ways. Oxidation can be applied to precondition organiccontaminants for biodegradation or be used as a polishing step to removeresidual contaminants (Brown, R. A., Nelson, C., Leahy, M. 1997.Combined oxidation and bioremediation for the treatment of recalcitrantorganics. In: B. C. Alleman and A. Leeson (Eds.). In Situ and On-siteBioremediation. Battelle Press, Columbus, Ohio, pp. 457-462). Ourresults showed that biodegradation followed by a modified Fentonreaction was more efficient than the reverse-order sequence. Theseresults suggest that easily biodegradable PAHs (i.e., two- andthree-ring compounds) were removed by biodegradation, and then, themodified Fenton's reagent appeared to target the more recalcitrant four-and five-ring PAHs. When a soil is dominated by four-ring to six-ringPAHs, chemical oxidation is best performed as a pretreatment step, andwhen two- and three-ring hydrocarbons are abundant, biodegradation isrecommended as the initial step to reduce excessive biodegradablehydrocarbons (Srivastava, V. J, Kelley, R. L., Paterek, J. R., Hayes, T.D., Nelson, G. L., Golchin, J. 1994. A field-scale demonstration of anovel bioremediation process for MGP sites. J. Appl. Biochem.Biotechnol. 45/46, 741-756). Indeed, in our MGP soil the concentrationof two- and three-ring hydrocarbons was about twice that of four- andfive-ring PAHs (Table 1).

Most studies conducted to date in application of a Fenton-type processhave focused on direct destruction of contaminants in soil, however, dueto the low pH requirement of the reaction for optimum efficiency, thereis cause for ecological concern. In the present study, we have developeda modified form of Fenton's reagent in an attempt to combine aFenton-type reaction with biodegradation. Hydroxyl radical has overtwice the oxidation potential of chlorine and is 25% stronger thanozone. In this study, biodegradation in conjunction with the powerfuloxidation potential of hydroxyl radicals was shown to enhance thedegradation of highly hydrophobic PAHs and to be more advantageous indestroying a mixture of PAHs than either treatment alone. In addition,the reagents used are inexpensive and the reactions are rapid andsimple. Furthermore, use of a chelating agent eliminates the concern ofan acidified soil environment which is associated with the originalFenton reaction. Our data demonstrate that combined biodegradation and amodified Fenton-type reaction is a promising technology to remediatesoils heavily contaminated with mixtures of PAHs.

The present invention will be of immediate relevance and applicabilityto the gas and electric utility industries and owners of former MGPsites with soils contaminated with coal tar residues. The technologycould also be applicable to wood treatment sites contaminated withcreosotes, to coke plant sites, to gas works sites contaminated with gascondensate residues, and to petroleum refineries and petroleum storagefacilities (such as tank farms) that have been contaminated with heavyoil fractions. The combined chemical and biological treatment processbeing investigated here could also be applied to treatment of mediacontaminated by other highly hydrophobic compounds, such as sedimentscontaminated with PCBs, chlorinated dioxins and dibenzofurans, or soilscontaminated with DNAPLs such as chlorinated solvents.

What is claimed is:
 1. A method of treatment of a contaminatecontaminated with an organic compound, comprising the steps of: (a)providing a contaminate, wherein the contaminate is contaminated with anorganic compound; (b) treating the contaminate with a chemical oxidationstep, wherein the chemical oxidation step comprises: contacting thecontaminate with a transition metal in soluble form; and an isolatedchelator of the transition metal, wherein the chelator of the transitionmetal and the transition metal form a transition metal:chelator complex;and an oxidizing agent that provides a reactive free radical in thepresence of the transition metal:chelator complex; and a buffering salt;wherein the pH is maintained in the neutral range; and wherein thereactive free radical initiates a chemical reaction with the organiccompound to produce reaction products of the organic compound; and (c)treating the contaminate with a bioremediation step, wherein thebioremediation step comprises contacting the contaminate with living,exogenous microbial consortium including at least two species underconditions suitable for the microbial consortium to mediatesolubilization or biodegradation of the organic compound or the reactionproducts thereof.
 2. The method of treatment according to claim 1,further comprising pre-treating the contaminate with the microbialconsortium prior to the chemical oxidation step.
 3. The method oftreatment according to claim 1, further comprising treating with themicrobial consortium alter the chemical oxidation step.
 4. A method oftreatment according to claim 1, wherein the contaminate is a particulatematerial, a surface soil, a subsurface soil, a sand, a silt a clay, asediment, a loam, a slurry, a colloid, a liquor, an industrial waterfluid, ground water, a pool, a pond or a lake.
 5. The method oftreatment according to claim 4, wherein the particulate material isgravel, pebbles, stone, stone chips, rock, ore, mining waste, coal,coke, slag, concrete, brick, construction material, demolition material,vermiculite, synthetic resin or plastic.
 6. The method of treatmentaccording to claim 1, wherein the contaminating organic compound isselected from the group consisting of a polycyclic compound, an aromaticcompound, a polycyclic aromatic compound, an aliphatic compound, anolefinic compound and an ethynic compound.
 7. The method of treatmentaccording to claim 6, wherein the contaminating organic compound is apolycyclic aromatic compound.
 8. The method of treatment according toclaim 7, wherein the polycyclic aromatic compound is selected from thegroup consisting of naphthalene, fluorene, phenanthrene, anthracene,pyrene, chrysene and benzo(a)pyrene.
 9. The method of treatmentaccording to claim 1, wherein the transition metal is manganese, iron,cobalt, nickel, copper or zinc.
 10. The method of treatment according toclaim 9, wherein the transition metal is iron.
 11. The method oftreatment according to claim 10, wherein the iron is present as ferrousiron, Fe(II), or ferric iron, Fe(III).
 12. The method of treatmentaccording to claim 11, wherein the iron is present as a perhalate. 13.The method of treatment according to claim 12, wherein the perhalate isa perchiorate, a perbromate or a periodate.
 14. The method of treatmentaccording to claim 13 wherein the perhalate is a perchlorate.
 15. Themethod of treatment according to claim 1, wherein the chelator of thetransition metal is an iron-chelator compound.
 16. The method oftreatment according to claim 15, wherein the iron-chelator compound is ahydroxylated benzene, a hydroxylated benzoic acid, a nitrilotriaceticacid, or a diethylenetriaminepentaacetic acid.
 17. The method oftreatment according to claim 16, wherein the hydroxylated benzene is adihydroxybenzene or a trihydroxybenzene.
 18. The method of treatmentaccording to claim 17, wherein the hydroxylated benzene is adihydroxybenzene.
 19. The method of treatment according to claim 18,wherein the dihydroxybenzene is catechol.
 20. The method of treatmentaccording to claim 16, wherein the hydroxylated benzoic acid is gallicacid.
 21. The method of treatment according to claim 16, wherein thehydroxylated benzoic acid is salicylic acid.
 22. The method of treatmentaccording to claim 1, wherein the pH is maintained in the range fromabout pH 5 to about pH
 8. 23. The method of treatment according to claim22, wherein the pH is maintained in the range from about pH 5.5 to aboutpH
 7. 24. The method of treatment according to claim 23, wherein the pHis maintained in the range from about pH 6 to about pH 6.5.
 25. Themethod of treatment according to claim 1, wherein the oxidizing agent isa peroxide.
 26. The method of treatment according to claim 25, whereinthe peroxide is hydrogen peroxide.
 27. The method of treatment accordingto claim 1, wherein the reactive free radical comprises an oxygenradical.
 28. The method of treatment according to claim 1, wherein theorganic compound of the contaminate is insoluble in aqueous solution.29. The method of treatment according to claim 1, wherein the reactionproducts are soluble in aqueous solution.
 30. The method of treatmentaccording to claim 1, wherein at least one of the reaction products ofthe organic compound is bioavailable tote microbial consortium.
 31. Themethod of treatment according to claim 1, wherein the oxidizing agentproduces an oxygen radical.
 32. The method of treatment according toclaim 1, wherein the buffering salt is a salt with a pKa in the neutralrange.
 33. The method of treatment according to claim 32, wherein thebuffering salt is a salt with a pKa in the range from about 5 to about8.
 34. The method of treatment according to claim 33, wherein thebuffering salt is calcium carbonate.
 35. The method of treatmentaccording to claim 1, wherein the molecular weight of the organiccompound of the contaminate is reduced.
 36. The method of treatmentaccording to claim 35, wherein the organic compound of the contaminateis substantially mineralized to carbon dioxide and water.
 37. The methodof treatment according to claim 1, wherein the organic compound of thecontaminate is toxic or carcinogenic to animals.
 38. The method oftreatment according to claim 37, wherein the organic compound of thecontaminate is toxic or carcinogenic to humans.
 39. The method oftreatment according to claim 1, wherein the reaction products arenon-toxic to animals.
 40. The method of treatment according to claim 39,wherein the reaction products are non-toxic to humans.
 41. The method oftreatment according to claim 1, wherein treating with the microbialconsortium yields at least one biodegradation reaction product, andwherein the products of biodegradation ore non-toxic to animals.
 42. Themethod of treatment according to claim 41, wherein the products ofbiodegradation are non-toxic to humans.
 43. The method of treatmentaccording to claim 41, wherein the biodegradation products are not knownto be carcinogenic in animals.
 44. The method of treatment according toclaim 43, wherein the biodegradation products are not known to becarcinogenic in humans.
 45. The method of treatment according to claim1, wherein the reaction products are not known to be carcinogenic inanimals.
 46. The method of treatment according to claim 45, wherein thereaction products are not known to be carcinogenic in humans.
 47. Themethod of treatment according to claim 1, wherein the organic compoundof the contaminate is from industrial manufacturing, industrialprocessing, chemical processing, coal tar processing, oil refining orenergy generation.
 48. The method of treatment according to claim 1,wherein the organic compound of the contaminate is from a naturalprocess.
 49. The method of treatment according to claim 1, wherein theorganic compound of the contaminate is halogenated.
 50. The method oftreatment according to claim 49, wherein the halogenated organiccompound of the contaminate is selected from the group consisting of atrichlorethene compound, a perchlorethene compound and apolychlorinated-biphenyl compound.
 51. The method of treatment accordingto claim 1, wherein the microbial consortium comprises at least two ofthe following: a bacterial species, a fungal species and an actinomycesspecies.
 52. The method of treatment according to claim 1, wherein themicrobial consortium is known to degrade the organic compound of thecontaminant.
 53. The method of treatment according to claim 1, whereinthe microbial consortium comprises at least two of the following: anAlcaligenes species, a Sphingomonas species, a Pseudomonas species, aRhodotorula species, a Burkholderia species, an Ochrobactrum species, aRhodococcus species, a Xanthomonas species and an Actinomyces species.